The soil bacterium Agrobacterium tumefaciens infects many species of dicotyledonous plants (dicots) and induces tumors called crown galls. The capacity of Agrobacterium to induce crown galls resides in its tumor-inducing (Ti) plasmid, which functions by transferring a region of the plasmid known as T-DNA into the host plant genome. The T-DNA is flanked by 24 base pair direct repeats which are required for transfer of the T-DNA to the host plant genome. The Agrobacterium plasmid T-DNA between the repeats can be deleted and replaced by genes of interest to provide a disarmed Agrobacterium useful for gene transfer into dicots.
Agrobacterium-mediated gene transfer is widely used for production of transgenic dicots. Generally, plant cells are transformed using Agrobacterium, and the transformed cells are regenerated into transgenic plants. Depending upon the plant species, the transformed cells may be derived from leaves, roots, hypocotyls, petioles, cotyledons, or seeds.
Transgenic dicots may also be obtained by an in planta transformation method, in which germinating seeds or wounded plants are inoculated with Agrobacterium, and plants are grown to maturity and seeds collected. An in planta transformation method has been used for Agrobacterium-mediated transformation of the dicot Arabidopsis thaliana. (Bechtold et al. (1993) Life Sci. 316:1194, C.R. Acad. Sci. Paris; Bechtold et al. in Gene Transfer to Plants, Potrykus et al., eds., Springer-Verlag Berlin, 1995, p. 19.) Vacuum infiltration with an Agrobacterium suspension was used to inoculate Arabidopsis plants at the panicle initiation stage. Plants were then transferred to soil, and seeds were collected from the inoculated plants. As many as ten independently-transformed plants can reportedly be derived from a single vacuum-infiltrated Arabidopsis plant. Bechtold et al., id., suggest that the success of the vacuum-infiltration transformation method as applied to Arabidopsis may be due to particular aspects of Arabidopsis biology, including the size of the plant, cycle length, and reproductive biology, which may prevent the method from being applied to other species.
Monocotyledonous plants (monocots) are generally less susceptible than dicots to Agrobacterium-mediated transformation, and thus direct DNA transfer methods have been widely used for monocot transformation. Direct DNA transfer methods include naked DNA uptake stimulated by polyethylene glycol or electroporation, and particle gun transformation. See, e.g., Gene Transfer to Plants, Potrykus et al., eds., Springer-Verlag, Berlin, 1995. For example, de la Pena (1987) Nature 325:272 report transformation by injection of DNA into floral tillers of rye plants. However, direct DNA transfer methods suffer deficiencies including frequent incorporation of the DNA into the host genome as multiple rearranged copies of the desired gene together with flanking sequences from the plasmid vector. These rearrangement and integration events may result in gene expression that is aberrant and unstable in R.sub.0 and progeny plants.
Agrobacterium-mediated gene transfer usually results in the insertion of a discrete, unrearranged DNA segment into the host genome, and thus it would be desirable to develop methods for the Agrobacterium-mediated transformation of monocots. Although monocots are considered to be relatively recalcitrant to transformation with Agrobacterium, there are various reports of gene transfer into monocots by Agrobacterium-mediated transformation (Boulton et al. (1989) Plant Mol. Biol. 12:31; Chan et al. (1992) Plant Cell Physiol. 33:577; Gould et al. (1991) Plant Physiol. 95:426; Graves et al. (1986) Plant Mol. Biol. 7:43; Grimsley et al. (1987) Nature 325:177; Raineri et al. (1990) Bio/Technology 8:33; U.S. Pat. No. 5,177,010 to Goldman et al. and U.S. Pat. No. 5,187,073 to Goldman et al.). However, many of these early studies of Agrobacterium-mediated transformation of monocots have been subject to controversy (Potrykus (1990) Bio/Technology 8:5350) and many have not been independently confirmed.
More recent studies report successful Agrobacterium-mediated transformation of rice. Hiei et al. (1994) Plant J. 6:271 disclose Agrobacterium-mediated transformation of Japonica rice. Various tissues from rice, including shoot apices, scutella, immature embryos, calli induced from young roots and scutella, and cells in suspension cultures induced from scutella were co-cultivated with A. tumefaciens, resulting in various levels of reporter transgene expression in these tissues. Transgenic plants were recovered from scutellum-derived calli that has been co-cultivated with A. tumefaciens. Stable integration, expression, and inheritance of transgenes was reported in R.sub.0, R.sub.1 and R.sub.2 generations.
U.S. Pat. No. 5,591,616 to Hiei et al. discloses a method for transforming a monocot comprising transforming a cultured tissue during the dedifferentiation process, or a dedifferentiated cultured tissue, with Agrobacterium. The cultured tissue is obtained by culturing an explant for not less than seven days on a dedifferentiation-inducing medium.
Dong et al. (1996) Molecular Breeding 2:267 report Agrobacterium-mediated transformation of Javanica rice. Co-cultivation of scutellar calli with A. tumefaciens resulted in transgenic fertile plants. The transgenes were transmitted to the R.sub.1 and R.sub.2 generations.
The foregoing methods of Agrobacterium-mediated transformation of rice involve the use of scutellar tissues or scutellar-derived callus, and involve relatively long-term in vitro selection procedures in tissue culture following the transformation process. Tissue culture manipulations have been associated with induction of somaclonal variation (Kaeppler et al. (1993) Proc. Natl. Acad. Sci. USA 90:8773; Phillips et al. (1994) Proc. Natl. Acad. Sci. USA 91:5222). Alternate methods of monocot transformation are desirable, for example to permit the use of tissue culture-recalcitrant cultivars and to reduce the incidence of somaclonal variations associated with the tissue culture process.
In accordance with the present invention, an Agrobacterium-mediated transformation method utilizing inflorescence of monocots as the target tissue has been developed.